IGEM:Caltech/2007/Protocols/cPCR

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Contents


Materials

  • template DNA or E. coli colonies
  • primer sets (3 µl; 10X stock)
  • PCR master mix (2X stock)
  • PCR grade water
  • buffer PB
  • buffer PE
  • buffer EB
  • spin column assemblies
  • collection tubes
  • 1.5 ml microfuge tubes
  • 0.2 ml thin-walled PCR tubes
  • benchtop microfuge
  • assorted pipetmen
  • assorted pipet tips
  • thermal block cycler
  • microcentrifuge
  • SpectraMax M2 spectrophotometer
  • toothpicks

Method 1: Conventional PCR

  1. Thaw the DNA template, the primer sets, and FastStart PCR Master Mix on ice. Label a 1.5 ml microfuge tube and a 0.2 ml PCR tube accordingly and place on ice.
  2. Set up a PCR program on the thermal block cycler as follows:
Cycles Cycles Time Temp
Initial Denaturation 1 4 min 95°C
Denaturation 101 30 sec 95°C
Annealing 30 sec 45-65°C1,3
Elongation 45 sec – 3 min 72°C
Denaturation 201 30 sec 94°C
Annealing 30 sec 45-65°C1,3
Elongation 45 sec – 3 min 72°C
Final Elongation 1 7 min 72°C
    1. If the PCR reaction involves no overhang regions, you can combine this two cycle sets into one set with 30 cycles. When the PCR reaction involves primers with overhangs (regions on the outside not complementary to the target), the first set of 10 cycles uses an annealing temperature based upon the part of the primers that are complementary to the target. The second set of 20 cycles uses an annealing temperature based upon the whole primer.
    2. Elongation time depends on the length of the amplification product. It is recommended to use approximately 1 minute per 1 kb of the PCR fragment.
    3. Exact annealing temperature depends on the melting temperature of the primers.

Save the program under “YOUR_NAME1” using times and temperatures appropriate for your amplification product and primers.

  1. Add the following reagents in the microfuge tube in the order listed with the appropriate pipetmen (note: the following is for a 50-l reaction):
forward primer (30 µM) 5 µl
reverse primer (30 µM) 5 µl
template DNA (10-250 ng) x µl
PCR grade water 15 – x µl

(up to 250 ng genomic DNA or 100 ng plasmid DNA/cDNA can be used)

  1. Mix the contents of the microfuge tube by flicking the tube contents or pipetting up and down with the L200 pipetman. Place the tube in the benchtop microfuge and pulse briefly to collect the volume at the bottom of the tube.
  2. Transfer the contents of this microfuge tube to the PCR tube with the P200 pipetman.
  3. Add 25 µl of the Master Mix to the PCR tube with the P200 pipetman. Mix by pipetting up and down with the L200 pipetman taking care to collect the volume at the bottom of the tube and to not introduce air bubbles into the reaction mixture. Place the PCR tube back on the ice.
  4. Return the Master Mix to the ice bucket (if done) or back to the -20ºC freezer. Transfer the PCR tube to the thermal block cycler and run the previously set up program.
  5. Once the program is complete remove the reaction from the thermal block cycler and store in your -20C until ready for subsequent purification.
  6. Go to method on PCR purification.

Method 2: Colony PCR

  1. Follow Steps 1 and 2 in Method 1.
  2. Add the following reagents in the PCR tube in the order listed with the appropriate pipetmen (note: the following is for a 25-l reaction):
forward primer (30 µM) 2.5 µl
reverse primer (30 µM) 2.5 µl
PCR grade water 7.5 µl
  1. Mix the contents of the PCR tube by pipetting up and down with the L200 pipetman.
  2. Using a toothpick, pick up a small amount of E. coli cells and place the toothpick cell-side in the PCR tube. Move the toothpick back and forth to disperse the cells. Throw away toothpick.
  3. Place the PCR tube in the thermal block cycler. Incubate the tube for 10 minutes at 90ºC.
  4. Remove the PCR tube from the thermal block cycle and add 25 µl of the Master Mix to the PCR tube with the P200 pipetman. Mix by pipetting up and down with the L200 pipetman taking care to collect the volume at the bottom of the tube and to not introduce air bubbles into the reaction mixture. Place the PCR tube back on the ice.
  5. Return the Master Mix to the ice bucket (if done) or back to the -20ºC freezer. Transfer the PCR tube to the thermal block cycler and run the previously set up program.
  6. Once the program is complete remove the reaction from the thermal block cycler and store in your -20C until ready for subsequent purification.
  7. Go to method on PCR purification.

PCR Purification

  1. Transfer your PCR mixture to an appropriately labeled 1.5 ml microfuge tube with the L200 pipetman.
  2. Add 5 volumes of buffer PB to the PCR sample (i.e., for a 50 µl reaction volume add 250 µl of buffer PB) with the L1000 pipetman. Mix the sample by pipetting up and down with the L1000 pipetman.
  3. Place a spin column into the collection tube and label appropriately. Transfer the entire PCR sample to the spin column assembly with the L1000 pipetman.
  4. Put the spin column assembly in the microcentrifuge, making sure to balance the tube weight with another spin column assembly or blank tube. Flow the PCR mixture through the spin column assembly by spinning at maximum speed for 1 minute.
  5. Remove the spin column assembly from the microcentrifuge. Remove the spin column from the collection tube and decant the filtrate from the collection tube into the sink. Place the spin column back into the collection tube.
  6. Transfer 750 µl of buffer PE to the spin column assembly using the L1000 pipetman.
  7. Put the spin column assembly back into the microcentrifuge, making sure to balance the tube weight with another spin column assembly or blank tube. Flow the wash buffer through the spin column assembly by spinning at maximum speed for 1 minute.
  8. Remove the spin column assembly from the microcentrifuge. Remove the spin column from the collection tube and decant the filtrate from the collection tube into the sink. Place the spin column back into the collection tube and return the assembly to the microcentrifuge, making sure the rotor is balanced.
  9. Dry the column membrane by spinning at maximum speed for 1 minute.
  10. Remove the spin column assembly from the microcentrifuge. Remove the spin column from the collection tube and transfer to an appropriately labeled 1.5 ml microfuge tube.
  11. Transfer 50 µl of buffer EB to the spin column with the L200 pipetman. Make sure to dispense the elution buffer directly on to the center of the spin column membrane, taking care to avoid contact with the wall of the column.
  12. Put the spin column assembly into the microcentrifuge, making sure to balance the tube weight with another spin column assembly or blank tube. Elute the PCR product from the spin column membrane by spinning at maximum speed for 1 minute.
  13. Remove the spin column assembly from the microcentrifuge. Remove the spin column from the microfuge tube and discard the spin column.
  14. The concentration of the eluted PCR product can be read by measuring the OD260 on the SpectraMax M2 spectrophotometer or the product can be analyzed on an agarose gel for size and specificity. Both of these methods are described in more detail in separate protocol handouts.
  15. The PCR product can be stored at -20C in your personal freezer box or used immediately in downstream applications.
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